Article pubs.acs.org/est
Aqueous Mesocosm Techniques Enabling the Real-Time Measurement of the Chemical and Isotopic Kinetics of Dissolved Methane and Carbon Dioxide Eric W. Chan,*,† John D. Kessler,† Alan M. Shiller,‡ DongJoo Joung,‡,§ and Frank Colombo∥ †
Department of Earth and Environmental Sciences, University of Rochester, Rochester, New York 14627, United States Department of Marine Science, University of Southern Mississippi, Stennis Space Center, Mississippi 39529, United States § Vermont EPSCoR, University of Vermont Burlington, Vermont 05405,United States ∥ Pactech Packaging, LLC, Rochester, New York 14624, United States ‡
S Supporting Information *
ABSTRACT: Previous studies of microbially mediated methane oxidation in oceanic environments have examined the many different factors that control the rates of oxidation. However, there is debate on what factor(s) are limiting in these types of environments. These factors include the availability of methane, O2, trace metals, nutrients, the density of cell population, and the influence that CO2 production may have on pH. To look at this process in its entirety, we developed an automated mesocosm incubation system with a Dissolved Gas Analysis System (DGAS) coupled to a myriad of analytical tools to monitor chemical changes during methane oxidation. Here, we present new high temporal resolution techniques for investigating dissolved methane and carbon dioxide concentrations and stable isotopic dynamics during aqueous mesocosm and pure culture incubations. These techniques enable us to analyze the gases dissolved in solution and are nondestructive to both the liquid media and the analyzed gases enabling the investigation of a mesocosm or pure culture experiment in a completely closed system, if so desired.
1. INTRODUCTION Blooms of microorganisms have been shown to be major removal mechanisms for chemical perturbations in aquatic systems, one of the more recent and notable examples being the Deepwater Horizon disaster in the Gulf of Mexico.1−5 A mesocosm is an experimental technique that involves collecting and observing a natural environmental sample (e.g., seawater) under controlled conditions and is ideal for a detailed study of such microbially mediated events.6−8 However, obtaining sufficient data during rapidly changing biogeochemical conditions, for example, during microbial blooms, is a common challenge that is pervasive even in more controlled laboratory experiments investigating pure cultures.9−11 This problem is especially challenging when investigating the biogeochemistry of dissolved gases; diffusive exchange with the atmosphere during sample storage and analysis can cause changes in dissolved gas concentration and the biogeochemical reaction. Here, we present techniques that do not contaminate the biogeochemical state of the system during incubation as well as techniques for the rapid, automated, and nondestructive analysis of dissolved greenhouse gas concentrations and stable isotopes. These techniques were developed with a focus on the biogeochemical formation and destruction mechanisms of methane (CH4), and thus, CO2 was also of interest as it is both a potential reactant and product for methane production and oxidation. However, these techniques can also be adapted to investigate different biogeochemical systems consuming or producing either of these greenhouse gases and other © XXXX American Chemical Society
accompanying constituents (e.g., trace metals, nutrients, dissolved oxygen, nitrogen species, other hydrocarbons, etc.). Previous investigations of methane oxidation have been limited by a paucity of techniques available for the time-course analysis of chemical and isotopic changes during microbial blooms. In several experiments, seawater is collected in glass vials and oxidation rates are determined only at a specific time rather than over the entire oxidation event; any time-course changes in concentrations and isotopes have only been measured at the beginning and end of the incubation due to the methodological limitations.12−14 One of the main challenges with incubating seawater in rigid vials is that the removal of an aliquot for analysis can often perturb the biogeochemical state by introducing a gaseous headspace, changing dissolved gas concentrations, and introduction of contaminants, thereby making the remainder of the sample useless for further analyses.1−3,514−17 Thus, the method we present here opens a new avenue of research because it enables for the first time the measurement of concentration and isotope changes over the entire course of a biogeochemical reaction especially with respect to methane kinetics. Prior studies into processes such as methane consumption (methanotrophy) and production (methanogenesis) have Received: September 4, 2015 Revised: January 26, 2016 Accepted: February 13, 2016
A
DOI: 10.1021/acs.est.5b04304 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. System setup with either mesocosm or pure culture configuration. (A) Mesocosm incubation system with samples bags inside protective cartridges. (B) Dissolved Gas Analysis System (DGAS) contains the Picarro G-2201i analyzer, valves to control liquid sample and gas flow, dissolved oxygen optode, temperature and pressure sensor, a mass air flow controller for purging the system. (C) The Weiss, ePTFE, and Liqui-cel equilibrators tested for use in the DGAS unit. (D) While our application presented is for discrete mesocosm samples, this system could be adapted for continuous analysis of a pure culture style experiment. (E) Control program written in National Instruments LabVIEW.
and dissolved gases analyzed can be returned back into the main sample thus providing a closed system setup that is nondestructive. This technique uses a laser spectroscopy analyzer that is capable of analysis at rates approaching 0.1 Hz, thus enabling very thorough investigations of dissolved gas chemical and isotopic dynamics during microbial blooms.
shown very interesting results with respect to chemical and isotopic kinetics during microbial blooms. For example, during the Deepwater Horizon disaster, several authors presented data suggesting a microbial bloom of methanotrophic bacteria completely oxidized the released methane within several months of the start of this disaster.1,2,4,18,19 However, this conclusion remains contentious due to suspicions of chemical limitations.5 A major uncertainty is how biogeochemical limitations affect the rates of aerobic methane oxidation during microbial blooms, highlighting the need for a more fundamental understanding of this biochemical process. Because the Deepwater Horizon methanotrophic response took place heterogeneously over an area of approximately 73 200 km2 in the northern Gulf of Mexico,2 such a fundamental understanding is difficult to obtain from the field data alone and more controlled mesocosm experiments are insightful. Similarly, stable isotopic kinetics have been previously investigated as a function of changing microbial growth during pure cultures of methanogenic archaea and bacteria.9−11 These studies observed changes in stable isotopes of methane and the isotopic fractionation factors as a function of microbial growth phase, varying as the cell growth continued through early phase, lag phase, and stationary phase growth. However, because the analytical techniques employed in these investigations were both manual and destructive, samples were collected at the gaseous exhaust of the bioreactor rather than the bioavailable methane dissolved in the media. In both of these previous examples, high temporal resolution measurements of chemical and isotopic changes would help quantify the biogeochemical dynamics during rapidly changing processes such as microbial blooms. Here, we present new high temporal resolution techniques for investigating dissolved gas concentration and stable isotopic dynamics during aqueous mesocosm and pure culture incubations. These techniques analyze the gases dissolved in solution and are nondestructive to both the liquid media as well as the analyzed gases enabling the investigation of a mesocosm or pure culture experiment in a completely closed system, if so desired. The analyzed aliquot
2. MATERIALS AND METHODS The techniques developed here involve three main independent and interconnecting parts (Figure 1). First, an incubation vessel was developed with the characteristics of being chemically clean, impermeable to dissolved gases, flexible to allow the removal of liquid without causing a vacuum or needing to introduce a substance for displacement, and structurally sound to hold a mass of water greater than 10 kg (Figure 1A; S1, Supporting Information). Second, because high precision and sensitivity analysis of methane and carbon dioxide can only be accomplished in the gaseous phase (not directly on gases dissolved in solution), a method of equilibrating the dissolved gas with the interior of a laser spectroscopy cavity was needed (Figure 1C). The laser spectroscopy technique employed here is Cavity-Ringdown Spectroscopy (CRDS; Picarro G2201-i) because it is high precision and nondestructive to the analyzed gases. Third, an integrated and automated system was developed for the clean, rapid, and automated analysis of samples in either a continuous closed-system analysis mode or a discrete sample mode. The integration of the second and third parts is labeled the DGAS unit (Dissolved Gas Analysis System) (Figure 1B). 2.1. Materials. 2.1.1. Sample Storage. Water samples are normally retrieved via precleaned Niskin or Go-flo bottles onboard a research vessel. Once these samples are brought back to the surface, they must be transferred to a storage vessel for the incubation experiment. This storage vessel must possess five characteristics: (1) The storage vessel must be chemically clean. While borosilicate glass vials are conventionally used for batch experiments,20 trace metals leaching from the glass and stoppers are a pervasive problem21,22 and could potentially influence the background metabolic activity of the microbial B
DOI: 10.1021/acs.est.5b04304 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology population.23 For this reason, acid washed polyethylene or Teflon bottles are generally used for trace metal sampling.21 (2) Because the biogeochemistry of dissolved gases is of interest, the vessel must have a low diffusivity for dissolved gases, a property that is true for glass but not for polyethylene and Teflon.24−27 (3) The vessel must allow for the removal of an aliquot for analysis without influencing the biogeochemical state of the system. To remove an aqueous aliquot from a sealed rigid vial, prior studies have either used displacement with an aliquot of pure gas or water or over pressuring the vials with a gaseous headspace.28 Unfortunately, these aliquot removal techniques perturb the biogeochemical state of the system, rendering further analyses of the dissolved gas dynamics useless. For this reason, a flexible vessel would be preferred so that the volume of the vessel would simply reduce as aliquots are removed. (4) Sample volumes of 10 L or more are not uncommon for mesocosm experiments,6 so the vessel must be structurally sound to support a mass of greater than 10 kg. (5) The vessel must be able to maintain the in situ pressure. Requirements 1−4 were satisfied using 15 L sample storage bags manufactured by Pactech Packaging LLC. The bags were custom-made from a laminated construction of five layers of film (outside to inside): polyester, low density polyethylene, metalized polyester, low density polyethylene, and linear low density polyethylene. The metalized polyester prevents diffusion of gas through the bag while the polyester and low density polyethylene provide a clean surface as well as structural integrity to the bags. These bags were held in protective cartridges constructed from 1/4 in. thick clear polycarbonate sheets. Each cartridge is fitted with Biochem pinch valves for controlling the flow of water from the bag without influencing the trace metal concentration (Figure 1A and Figure S1). While maintaining the in situ pressure would be preferred, this cannot be accomplished outside of the ocean without sacrificing requirements 1 and 3. Thus, requirement 5 was ignored. Once samples are ready for incubation, they are placed into the custom incubator. The cartridges that house the sample bags act as an extra layer of protection for the bags as well as a storage solution inside the incubator (Figure 1A; Figures S1 and S2). The incubator consists of a 700 L Frigidaire freezer controlled to subambient temperatures above freezing with a custom proportional integral derivative (PID) controller (Omega CNi-3253). Temperature precisions of
